Project Summary/Abstract Down syndrome (DS) is the most common cause of intellectual disabilities, congenital heart defects and early-onset Alzheimer?s disease. It occurs at a frequency of almost 1 in 700 live births, and is due to the complete or partial trisomy of human chromosome 21 (Hsa21). However, the specific molecular mechanisms giving rise to DS pathologies remain elusive. Mitochondrial dysfunction and oxidative stress are widely reported in cells and tissues from DS subjects and are thought to be important contributors to the early-aging, degenerative nature of DS. Our laboratory recently reported that the mitochondrial network in induced pluripotent stem cells (iPSCs) derived from DS patients (T21) is more fused and metabolically active than the mitochondrial population in isogenic, diploid iPSCs. This remarkable finding suggests that, on a fundamental level, DS mitochondria may be super-functional rather than dysfunctional. We went on to show that siRNA depletion of Regulator of Calcineurin 1 (RCAN1/DSCR1), a gene on Hsa21, is sufficient to restore a more normal mitochondrial morphology and function to T21 iPSCs. RCAN1 was previous identified by our lab as a feedback inhibitor of the Ca2+-activated protein phosphatase calcineurin. Calcineurin-mediated dephosphorylation of Drp1, an important regulator of mitochondrial dynamics, promotes mitochondrial fission, a process that tends to decrease mitochondrial metabolic activity but that is essential for turnover and repair of damaged mitochondria. We postulate that increased dosage of RCAN1 in DS suppresses the process of calcineurin-mediated mitochondrial fission. Our underlying hypothesis is that elevated metabolic activity and ROS production, coupled with a decreased capacity to remove damaged mitochondria, increases cumulative, oxidative damage over time in individuals with DS. Our proposed studies will (Aim 1) Test this hypothesis at the level of early stem cell differentiation and cell fate commitment by assessing changes in mitochondrial function and indices of cellular damage during differentiation of isogenic triploid T21 and diploid D21 iPSCs. (Aim 2) Use the powerful tool of CRISPR-Cas9 genomic editing to test whether trisomy of RCAN1, or other loci, is sufficient to drive the hyper-fused phenotype, and (Aim 3) Explore possible therapeutic approaches to restoring normal mitochondrial function using newly identified compounds capable of preventing RCAN1 from inhibiting calcineurin. These studies address an important gap in our knowledge regarding the molecular causes of DS. Identifying the dominant genes or molecular pathways involved would open the possibility for developing targeted therapies to improve the lives of affected individuals and their families. In addition, many of the pathologies that occur with high frequency in DS are also common in the general population, although at a lower frequency, such as cardiac cushion defects, patent ductus arteriosus, diabetes, and Alzheimer?s disease. Thus, a better understanding of the specific mechanisms contributing to the DS phenotype is likely to yield insights that will also benefit a much wider population.